Molecular biology of enzyme adaptations in higher eukaryotes

Multicellular eukaryotes have evolved complex homeostatic mechanisms that buffer the majority of their cells from direct interaction with the external environment. Thus, in these organisms long-term adaptations are generally achieved by modulating the developmental profile and tissue specificity of gene expression. Nevertheless, a subset of eukaryotic genes are still involved in direct responses to environmental fluctuations. It is the adaptative responses in the expression of these genes that buffers many other genes from direct environmental effects. Both microevolutionary and macroevolutionary patterns of change in the structure and regulation of such genes are illustrated by the sequences encoding alpha-amylases. The molecular biology and evolution of alpha-amylases in Drosophila and other higher eukaryotes are presented. The amylase system illustrates the effects of both long-term and short-term natural selection, acting on both the structural and regulatory components of a gene--enzyme system. This system offers an opportunity for linking evolutionary genetics to molecular biology, and it allows us to explore the relationship between short-term microevolutionary changes and long-term adaptations.     

Mitochondrial DNA reveals a mosaic pattern of phylogeographical structure in Atlantic and Mediterranean populations of anchovy (Engraulis encrasicolus)

This study extends the geographic coverage of a previous study of mitochondrial DNA restriction fragment length polymorphism in European anchovy. Both studies together include 24 samples representing 17 localities extending from the Black Sea, through the Mediterranean Sea to the eastern Atlantic as far south as Dakar, Senegal. Eighty-eight haplotypes define two clades (A and B) separated by 3.2% sequence divergence. Clade A has a star-like genealogy indicative of a recent population expansion. Clade B has a more complex genealogy, consisting of several haplotypes at intermediate frequencies. The distributions of these clades consist of a mosaic with abrupt changes between some areas and gradients between other areas. Clade A predominates the Black and Aegean seas, but is present throughout the Mediterranean. Unexpectedly, new data show that clade A is also at a high frequency in the Atlantic, from Portugal to at least Senegal. Overall, the level of genetic differentiation among populations is high (F(ST)=0.148, p<0.0001), with the greatest differences between basins. AMOVA reveals four main geographical groups: Atlantic, central Mediterranean, Aegean Sea, and Black Sea. Mismatch distribution clearly indicates historical bottleneck and population expansion for clade A, while for clade B such evidence is equivocal. This difference may reflect a range expansion for both clades, but with higher gene flow (Nm values) between demes for clade A. Both contemporary and historical processes are important in shaping the complex genetic population structure of European anchovy.     

Synthesis and evaluation of the antioxidative potential of minoxidil–polyamine conjugates

A series of conjugates (MNX–CO–PA) of minoxidil (MNX) with the polyamines (PAs) putrescine (PUT), spermidine (SPD) and spermine (SPM) as well as dopamine were produced through activation of MNX with N,N′-carbonyldiimidazole, followed by reaction with dopamine or selectively protected PAs and acid-mediated deprotection. These conjugates together with conjugates of the general type MNX–PA or PA–MNX–PA, readily produced using literature protocols, were tested as antioxidants. The most potent inhibitors of lipid peroxidation were the conjugates MNX–SPM (2, 94%), SPM–MNX–SPM (4, 94%) and MNX–N⁴-SPD (7, 91%) and MNX (91%). The most powerful lipoxygenase (LOX) inhibitors were MNX (IC₅₀ = 20 μM) and the conjugates MNX–N⁸-SPD (9, IC₅₀ = 22.1 μM), MNX–CO–dopamine (11, IC₅₀ = 28 μM) and MNX–N¹-SPD (8, IC₅₀ = 30 μM). The most interesting conjugates 2, MNX–CO–PUT (5), 8 and 11 as well as MNX were generally found to exhibit weaker (22–36.5%) or no (conjugate 8) anti-inflammatory activity than indomethacin (47%) with the exception of MNX which showed almost equal potency (49%) to indomethacin. The cytocompatibility of conjugates and MNX at the highest concentration of 100 μM showed a survival percentage of 87–107%, with the exception of conjugates with SPM (compound 2) and MNX–CO–SPM (6), which showed considerable cytotoxicity (survival percentage 8–14%). Molecular docking studies were carried on conjugate 9 and the parent compound MNX and were found to be in accordance with our experimental biological results.     

Assessing the genetic landscape of a contact zone: the case of European hare in northeastern Greece

The European hare populations of the Balkan Peninsula comprise two divergent phylogenetic lineages with discrete geographical distribution slightly overlapping in the area of northeastern Greece and Bulgaria. Here we elucidate their contact zone, by defining the spatial distributional pattern of the two highly divergent groups, detecting individuals of hybrid origin, and identifying genetic barriers present in the area of their co-existence. Specimens from northeastern Greece were assayed for lineage assignment and population genetic inference based on a 511 bp fragment of mitochondrial DNA control region and allelic data from 10 microsatellite loci. Bayesian analyses on original and simulated genotypes were performed allowing for the contact zone delineation. Our results indicate high genetic diversity in both nuclear and mitochondrial DNA, strong population structure and non random spatial distribution of the differentiated gene pools. The information provided by the two types of molecular markers yielded consistent results. This study comprises a fine scale analysis of the contact zone between the two evolutionary lineages of European brown hares in northeastern Greece. Specific questions on the spatial patterns where addressed for the first time. Furthermore, hypotheses regarding the presence of hybrids were also tested. As a result, interpretive power to the diversity patterns observed today in the Balkans was added and previously overlooked aspects of the species biology were highlighted.     

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.     

New polymorphic microsatellite loci for the Greek smooth newt, Lissotriton vulgaris graecus, and their utility in the nominotypical subspecies

Eight polymorphic microsatellites were isolated from the Greek smooth newt (Lissotriton vulgaris graecus) using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC)(11) probe. The loci showed different variation patterns in a single breeding population (32 individuals) with mean number of alleles at 5.0 and mean observed heterozygosity at 0.520. The amplification success also in the nominotypical subspecies favours the use of these microsatellite loci in population genetic analyses as well as in the study of contact zones between smooth newt subspecies.     

The imbalanced supertree of flowering-plant phylogeny

Two contrasting approaches have been used to construct the overall tree of life from molecular data: one involves the analysis of single large datasets, while the other involves joining many independent smaller analyses into a supertree. A recent study uses the latter approach to produce the most complete phylogeny yet of flowering plant families.     

Recovery of phenolic antioxidants from wine industry by-products

The recovery process of the phenolic compounds contained in the wine industry by-products and their antioxidant activity were examined in this work. To this purpose, a combined process of liquid and supercritical solvent extraction was employed. At first the effect of various process parameters of the liquid solvent extraction--the type of solvent and the pretreatment of the raw material (composition (skins, seeds, stems) and crushing)--on the antioxidant activity of the extract was examined. It was shown that an extract of a higher antioxidant activity was obtained by using ethyl acetate as solvent and raw material free of stems. These extracts were further treated with supercritical carbon dioxide (SC CO(2)) at various extraction pressures, which resulted in their significant enrichment in phenolic compounds and the improvement of their antioxidant and organoleptic properties, especially at pressures higher than 100 bar. The antioxidant activity of the products was determined by using the Rancimat method, as well as a simple and not time-consuming free radical method. It was proved that both the ethyl acetate extracts and those treated with SC CO(2) had antioxidant activity comparable to that of antioxidants commonly used in industry, that is butylhydroxytoluene (BHT), a synthetic antioxidant, and Rosemary extract, a widely known natural one.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey